Tryptophan hydroxylase (TPH) catalyzes the initial and rate-limiting step in the biosynthesis of serotonin. As such, it has been implicated in a variety of mental health disorders. One of the goals of this long-standing R01 has been to better understand this pivotal enzyme. However, in the midst of the preceding cycle, other investigators (and current collaborators) made the seminal discovery of a new variant of this enzyme (encoded by a separate gene) that is responsible for central nervous system serotonin synthesis - TPH2. Given that the work in this field had to this point focused on the peripheral enzyme (now termed TPH1), and given the importance of CNS serotonin to health and disease, we propose shifting the emphasis of this upcoming renewal period to better understanding this novel and pivotal enzyme. Virtually nothing is known about the structure, function, and enzymology of hTPH2. Partnering with the discoverers of this new enzyme, we have established important new characterization studies on the human TPH2 (hTPH2). We are strongly positioned to pursue three specific aims in the present proposal. Specific Aim 1 will characterize the functional consequences of naturally-occurring polymorphisms in the hTPH2 gene. In the few short years since discovery of the TPH2 gene, seven different coding region polymorphisms have been described. Little is known concerning the consequences of these amino acid substitutions and this aim will address this gap in our knowledge. Aim 2 will use data we have obtained from tyrosine hydroxylase and hTPH1 to map the active site of hTPH2. These biochemical studies will provide important functional insights into this novel and important enzyme. Specific Aim 3 will explore the regulation of hTPH2 by its N-terminal regulatory domain. These experiments will contribute to our knowledge of the dynamic regulation of serotonin biosynthesis in health and disease. The novel hTPH2 gene was first described three years ago. Its discovery resolves several important discprepancies in the field of serotonin biosynthese. However, we must establish a deeper understanding of this pivotal enzyme to better appreciate its role in health and disease and to provide a basis for potential development of novel pharmacotherapeutics.